Compositions and methods for assaying the activity of S-adenosylmethionine (SAM)-dependent methyltransferases and for screening for modulators of the activity SAM-dependent methyltransferases are provided. Kits and articles of manufacture that contain the modified enzymes and that can be used in these methods are also provided herein.
Of the 3,196 enzymes described in the latest version of Enzyme Nomenclature (Webb, Enzyme Nomenclature, Academic Press, San Diego (1992)), about 3% represent species that catalyze the attack of a variety of nitrogen, oxygen, carbon, and sulfur nucleophiles on the methyl group of S-adenosylmethionine (Kagan and Clarke, Arch. Biochem. Biophys., 310(2):417-427 (1994)). These methyltransferases include enzymes that result in the formation of methyl esters, methyl ethers, methyl thioethers, methyl amines, methyl amides and other derivatives on proteins, nucleic acids, polysaccharides, lipids and various small molecules. Among these methyltransferases are S-adenosylmethionine (SAM)-dependent methyltransferases.
Methyltransferase, including SAM-dependent methyltransferase, catalyzed abnormal methylation has been linked to pathological conditions (see, e.g., U.S. Pat. No. 5,876,996). For example, covalent modification of cellular substrates with methyl groups has been implicated in the pathology of cancer and other diseases (Gloria, et al., Cancer, 78:2300-2306 (1996)). Cytosine hypermethylation of eukaryotic DNA prevents transcriptional activation (Turker and Bestor, Mutat. Res., 386:119-130 (1997)). N6-methyladenosine is found at internal positions of mRNA in higher eukaryotes (Bokar, et al., J. Biol. Chem., 269:17697-17704 (1994)). Hypermethylated viral DNA is transcribed at higher rates than hypo- or hemimethylated DNA in infected cells (Willis, et al. Cell. Biophys., 15:97-111 (1989)).
In addition, many pathways of small molecule degradation, such as those of neurotransmitters, require methyltransferase activity (U.S. Pat. No. 5,876,996; and Kagan and Clarke, Arch. Biochem. Biophys., 310:417-427 (1994)). Degradation of catecholamines (epinephrine ornorepinephrine) requires phenylethanolamine N-methyltransferase. Hydroxyindole methyltransferase converts N-acetyl-5-hydroxytryptarnine to melatonin in the pineal gland.
In their roles as a rate-limiting step in methyltransferase reactions, SAM-dependent methyltransferases have been identified as targets for psychiatric, antiviral, anticancer and anti-inflammatory drug design (U.S. Pat. No. 5,876,996). For instances, sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions (Minarovits et al., Virology, 200:661-667 (1994)). 2xe2x80x2-5xe2x80x2-linked oligo(adenylic acid) nucleoside analogues synthesized by interferon-treated mouse L cells act as antiviral agents (Goswarmi, et al., J. Biol. Chem., 257:6867-6870 (1982)). Adenine analog inhibitors of AdoMet-MT decreased nucleic acid methylation and proliferation of leukemia L1210 cells (Kramer et al., Cancer Res., 50:3838-3842 (1990)).
Hence, methyltransferase activity can be associated with certain pathologies and other conditions, and its acitivity used as an indicator of diseases and conditions. Available methods for assessing the activity of methyltransferases, including SAM-dependent methyltransferases, require the use of radioactively labelled reagents and/or sophisticated and expensive analytical equipment such as HPLCs and GC/MS. To fully exploit the diagnostic potential of the methyltransferase activity as a diagnostic aid, more convenient and cost-effective methods for assessing activity are required.
Therefore, it is an object herein to provide quick and simple methods for assaying the methyltransferase, such as SAM-dependent methyltransferase, activity. It is also an object herein to provide for methods for screening for modulators of such methyltransferases, including SAM-dependent methyltransferases.
Provided herein are methods for assessing the activity of methyltransferases. The methods are exemplified with references to SAM-dependent methyltransferase, but can be applied to any transferase. In particular, methods for assaying the activity of a S-adenosylmethionine (SAM)-dependent methyltransferase are provided. The methods are practiced by contacting the SAM-dependent methyltransferase with a substrate of the methyltransferase in the presence of a methyl donor, such as SAM, whereby a methyl group is transferred from donor, SAM, to a substrate for the methyltransferase. In the instance where SAM is the donor, SAM is converted to S-adenosylhomocysteine (SAH), which is a demethylated donor. The resulting product, in this instance SAH, is contacted with an mutant binding enzyme, in this instance a mutant SAH-binding enzyme, which has binding affinity for SAH (or other product) but has attenuated catalytic activity. The binding between the mutant enzyme and the product, in this instance SAH is detected, and correlated with the amount of product, SAH, which in turn is correlated with the level of activity of the methyltransferase that generated the product.
This reaction, exemplified with reference to SAM-dependent methyltransferase, is summarized as follows:
R is selected from proteins, DNA, RNA, lipids and small molecules. 
Hence, a reaction scheme, such as the above, can be used to screen for compounds that modulate the activity of the selected methyltransferase. Modulators include inhbitors, which could serve, for example, as anticancer agents, anti-neurodegenerative agents, and anti-inflammatory agents.
Hence performance of the methods provided herein requires a methy donor, and a mutant enzyme that binds to the demethylated donor, but does not catalyze further reaction thereof. Methods of use and preparation of such mutant binding enzymes are described herein and also described and exemplified in copending U.S. application Ser. No. 09/347,878 to Chong-Shen Yuan, filed Jul. 6, 1999, entitled xe2x80x9cCOMPOSITIONS AND METHODS FOR ASSAYING ANALYTESxe2x80x9d and U.S. application Ser. No. 09/457,205 to Chong-Shen Yuan, filed Dec. 6, 1999, entitled xe2x80x9cCOMPOSITIONS AND METHODS FOR ASSAYING ANALYTES.xe2x80x9d U.S. application Ser. No. 09/457,205, which are incorporated by reference in their entirety.
Also provided herein, are methods for identifying modulators of methyltransferases. The methods assess methyltransferase activity by detecting binding of a mutant binding enzyme to the demethylated methyl donor in the methyltransferase reaction. The reactions are performed in the presence of a test compound, and any change (relative to a control) in the amoung mutant enzyme-demethylated product form is indicative of an effect of the test compound on the methyltransferase. Typically the control is the amount of binding in the absence of the test compound or comparison with any suitable standard curve prepared for this purpose. Compounds that result in an altered amount of mutant binding enzyme-methyl donor complex compared to a suitable control are selected for further screening.
The exemplified methods are described with reference to SAM-dependent methyltransferase using SAM as the methyl donor, and SAH as the demethylated product that forms a complexewith the mutant enzyme. In an exemplary embodiment the methods for identifying modulators of methyltransferase activity are practiced by contacting the SAM-dependent methyltransferase with a substrate of the methyltransferase in the presence of SAM and a test substance, whereby a methyl group is transferred from SAM to the substrate and SAM is converted to S-adenosylhomocysteine (SAH); contacting the SAH with a mutant SAH-binding enzym that has binding affinity for SAH but has attenuated catalytic activity, and detecting binding between SAH and the mutant SAH-binding enzyme to detect or determine the presence or amount of SAH, whereby the activity of the SAM-dependent methyltransferase is assessed; and comparing the assessed activity of the SAM-dependent methyltransferase with a control.
If the activity measured differs from the activity measured in a control reaction, such as the same reaction performed in the absence of the test compound or a standard curve or other measurement whereby the aunmodulated activity of the methyltransferase can be assessed, then the test substance modulates the activity of the methyltransferase, such as the SAM-dependent methyltransferase.
In certain embodiments, the control is performed by contacting the SAM-dependent methyltransferase with a substrate of the methyltransferase in the presence of SAM, whereby a methyl group is transferred from SAM to the substrate and SAM is converted to S-adenosylhomocysteine (SAH), contacting the SAH with a mutant SAH-binding enzyme that has binding affinity for SAH but has attenuated catalytic activity, and detecting binding between SAH and the mutant SAH-binding enzyme to detect or determine the presence or amount of SAH, whereby the activity of the SAM-dependent methyltransferase is assessed.
Further provided herein are compositions, kits and articles of manufacture to be used in the above assay or screening methods.
The methyltransferases that can be assayed or screened against in the above methods include, but are not limited to, protein methyltransferases, nucleic acid methyltransferases, lipid methyltransferases, polysaccharide methyltransferases and other small molecule methyltransferases. Particularly preferred are SAM-dependent methyltransferases.
The methyltransferase can be isolated from a target cell. Hence in a specific emdodiment, the methyltransferase is a SAM-dependent methyltransferase that is isolated from a target cell, the test substance to be screened for is a therapeutic compound, and the screening assay is used to assess whether the target cell responds to the test substance
For use with the SAM-dependent methyl transferase the mutant enzyme binds to the demethylated product SAH. In a preferred embodiment, the mutant SAH-binding enzyme used is a mutant SAH hydrolase, which mutant SAH hydrolase substantially retains its binding affinity or has enhanced binding affinity for SAH but has attenuated catalytic activity. In another preferred embodiment, the assay or screening is used in a high throughput mode, i.e., the activities of a plurality of SAM-dependent methyltransferases are assayed simultaneously and/or a plurality of test substances are screened for simultaneously.